US6875580B2 - Antibodies specific for plasmacytoid dendritic cells - Google Patents

Antibodies specific for plasmacytoid dendritic cells Download PDF

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US6875580B2
US6875580B2 US10/764,718 US76471804A US6875580B2 US 6875580 B2 US6875580 B2 US 6875580B2 US 76471804 A US76471804 A US 76471804A US 6875580 B2 US6875580 B2 US 6875580B2
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cells
pdc
cd11c
antibody
mice
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US20040166108A1 (en
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Carine Paturel
Giorgio Trinchieri
Jean-Jacques Pin
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Merck Sharp and Dohme LLC
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Schering Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells

Definitions

  • the invention provides immunological reagents (antibodies) capable of binding to plasmacytoid dendritic cells (pDC), to cell lines which express such antibodies and to a process for identifying and purifying plasmacytoid dendritic cells from tissues containing pDC using such antibodies.
  • immunological reagents capable of binding to plasmacytoid dendritic cells (pDC)
  • pDC plasmacytoid dendritic cells
  • DC Dendritic cells
  • APC antigen-presenting cells
  • pDC plasmacytoid DC
  • Ig-secreting plasma cells Grouard et al., 1997 , J. Exp. Med. 185(6):1101-1111
  • unique surface phenotype CD4+IL-3R++CD45RA+HLA-DR+
  • pDC are believed to be derived from a precursor common with T cells and B cells (Grouard et al., 1997 , J. Exp. Med. 185, 6:1101-1111; Res et al., 1999 . Blood 94, 8:2647-57; Res et al., 1999 , Blood 94 (8):2647-57; Bruno et al., 1997 , J. Exp. Med. 185:875-884; Bendriss-Vermare et al., 2001, JCI 107 :835; Spits et al., 2000 , J. Exp. Med. 192 (12):1775-84).
  • Mouse pDC have been recently identified by several groups as CD11c low B220 hi Gr1 low cells, able to produce type I IFN in response to viral stimulation and exhibiting plasmacytoid morphology (Nakano et al., 2001 , J. Exp Med, 194(8):1171-8 ; Paturel et al., 2001 , Nat Immunol. 2(12):1144-1150; Bjorck, 2001 , Blood 98(13):3520-6). Mouse pDC can also be obtained in large number in vitro, by differentiating bone marrow cells into dendritic cells in the presence of FLT3L.
  • pDC In addition to their morphology, their IFN ⁇ production and their putative origin, pDC also differ from myeloid DC in their weak phagocytic activity (Grouard et al., 1997 , J. Exp. Med. 185, 6:1101-1111), their weak IL-12 production capacity (Rissoan et al., 1999 , Science 283:1183-1186), and the signals inducing their activation (Kadowaki et al., 2001 , J. Immunol 166(4):2291-5).
  • pDC have been shown to induce IL-10 secreting T cells (Rissoan et a., 1999 , Science 283:1183; Liu et al., 2001 , Nature Immunol 2:585) and CD8 regulatory T cells (Gilliet et al., 2002 , J. Exp Med. 195(6):695-704).
  • Human natural IFN-producing cells HuIPC
  • NK natural killer
  • pDC have been recently associated with auto-immune diseases, in particular Lupus erythematosus (Farkas et al., 2001 , Am. J. Pathol. 159(1)237-43).
  • Type I interferons are central players in host resistance to viral or microbial infections (Pfeffer et al., 1998 , Cancer Res 58(12):2489-99; van den Broek et al., 1995 , Immunol Rev 69(8):4792-6).
  • the critical role of pDC in viral infection has been recently demonstrated in vivo, in MCMV and VSV infection models (Dalod et al., 2002 , J Exp Med 195(4):517-28; Barchet et al., 2002 , J Exp Med 195(4):507-16).
  • mice infected with MCMV the level of IFN ⁇ is dramatically decreased in mice infected with MCMV.
  • the anti-Gr1 treatment used to deplete pDC could in addition to neutrophils, also possibly deplete a proportion of macrophages and of activated T cells.
  • the present invention fills the foregoing need by providing a binding compound having the binding characteristics of a monoclonal antibody produced by the hybridoma cell line deposited on Jan. 27, 2003 under ATCC Accession No. PTA-4957.
  • the binding composition is an antibody or antibody fragment.
  • the monoclonal antibody is monoclonal antibody 120G8 produced by hybridoma ATCC No. PTA-4957.
  • hybridoma cell line having Accession No. ATCC PTA-4957.
  • the invention further provides methods for purifying pDC from a sample containing pDC, which method comprises contacting said sample with a 120G8 antibody and then recovering pDC which have bound to said antibody.
  • the invention provides a method for identifying pDC from a sample containing pDC comprising the steps of contacting said sample with a 120G8 antibody to form an antibody/pDC complex, and detecting the presence of said antibody/pDC complex to identify the pDC.
  • the present invention is based, in part, on the discovery of an antibody which specifically recognizes pDC in the mouse.
  • T cell response upon presentation of antigen by DC is dependent on the subpopulation of DC involved and the stage of maturation of presenting DC (Steinman et al., 2000 , J. Exp. Med. 191(3):411-6).
  • MDC and pDC are able to polarize the type of the T cell response toward a Th1 or a Th2 response through their capacity to secrete IL-12 or not, respectively (Rissoan et al., 1999 , Science 283:1183-1186).
  • the two DC subtypes also make different links between acquired and innate immune responses, with MDC activating both B cells (Dubois et al., 1999 , J. Leukoc. Biol.
  • This antibody which is of the IgG1/ ⁇ isotype, has been designated 120G8.
  • the 120G8 antibody is produced by a hybridoma cell line that was generated by the fusion of spleen cells with the murine myeloma cell line SP 2 O. This hybridoma was deposited on Jan. 27, 2003 under the Budapest Treaty as ATCC Accession No. PTA-4957. The American type Culture Collection ATCC, is located at 10801 University Boulevard., Manassas, Va. 20110-2209 in the United States of America.
  • the 120G8 mAb can be used to selectively isolate pDC from total cells. It stains pDC from either ex vivo total cells or in vitro bone marrow-derived DC.
  • pDC originating not only from different organs in the mouse, but also from different mice strains. It can be used in fluorescence activated coil sorter (FACS) studies, immunohistochemistry (IHC) or immunohistofluorescenve (IHF) staining on tissue sections. Because 120G8 mAb recognizes both resting and activated pDC, it is most helpful to study pDC response to activation, in vitro and in vivo. Finally 120G8 mAb injection in vivo depletes mice of pDC, as determined both phenotypically and functionally.
  • FACS fluorescence activated coil sorter
  • IHC immunohistochemistry
  • IHF immunohistofluorescenve
  • binding compound as used herein includes antibodies and functional fragments thereof which specifically bind pDC, and which have an epitopic specificity which is the same as or similar to that of the 120G8 mAb described herein. Binding compounds which have an epitopic specificity which is the same as or similar to that of 120G8 mAb can be identified by their ability to compete with 120G8 mAb for binding to pDC.
  • BALB/cByJ Specific pathogen-free BALB/cByJ, 129 SvPas, C57BI/6J, CBA/J, C3H/HeN, DBA/2J, BALB/c-nude female mice, 6-8 weeks of age, were purchased from Charles River (Iffa-Credo, L'Arbresle, France). All mice experiments were performed following protocols approved by the institutional animal committees and in accordance with EEC Council Directive 86/609 as well as institutional animal care and use guidelines.
  • RPMI 1640 (Life Technologies, Paisley Park, U.K.) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS, Life Technologies), 2 mM L-Glutamine (Life Technologies), 80 ⁇ g/ml Gentallin (Schering Plough, Union, N.J.), 10 mM Hepes (Life Technologies), 50 ⁇ M , ⁇ 2-mercaptoethanol (Sigma, St Louis, Mo.), at 37° C. in 5% CO 2 .
  • High density supernatants of hybridoma were produced in DMEM/F12 (Life Technologies), supplemented with, 2 mM L-Glutamine, 80 ⁇ g/ml Gentallin. 10% (v/v) horse serum (HS, Life Technologies) was added unless specified. All antibodies were from Pharmingen (San Diego, Calif.), unless otherwise specified.
  • PBS-FCS-EDTA PBS (Life Technologies) supplemented with 5% (v/v) heat-inactivated FCS and 0.5 mM EDTA (Sigma). Blood cells were collected in excess PBS
  • T and B cell depletions cells were incubated for 30 min at 4° C. with a mixture of anti-CD3 molecular complex (17A2), anti-CD8 ⁇ (53-5.8), anti-CD19 (1D3) or anti-erythrocyte (TER119) were alternatively used.
  • Cells and goat anti-rat IgG-coated Dynabeads (Dynal, Oslo, Norway) were mixed under continuous agitation for 15 min at 4° C. Beads and attached cells were removed using a Dynal magnet.
  • CD11c + cells were purified by positive selection using CD11c + Microbeads and MiniMacs (Myltenyi Biotec, Bergisch Gladbach, Germany) starting from either total spleen cells, total bone marrow cells or CD19, CD3, CD8 ⁇ , TER119 depleted cells.
  • BM-DC bone marrow in vitro derived-DC
  • FLT3L bone marrow in vitro derived-DC
  • isolated bone marrow cells plated at 10 6 cells/ml in 24-well plates were incubated for 9 days in complete RPMI1640 medium supplemented with 25 ng/ml recombinant murine FLT3L (R&D systems, Abingdon, U.K.). Medium was renewed every 2-3 days.
  • Mouse spleen cells from BALB/c mice were incubated for 30 min at 4° C. with a mixture of rat mAb including anti-CD3 molecular complex (17A2), anti-CD8 ⁇ (53-5.8), anti-CD19 (1D3), anti-CD5 (53-7.3), anti-CD11b (M1/70), and anti-erythroc (TER119) then antibody-coated cells were removed using Dynabeads.
  • rat mAb including anti-CD3 molecular complex (17A2), anti-CD8 ⁇ (53-5.8), anti-CD19 (1D3), anti-CD5 (53-7.3), anti-CD11b (M1/70), and anti-erythroc (TER119) then antibody-coated cells were removed using Dynabeads.
  • Depleted cells were stained with rat anti-Ly6G/C (RB6-8C5)-phycoerythrin (PE), hamster anti-CD11c (HL-3)-biotin, and a cocktail of fluorescein isothiocyanate (FITC)-labeled hamster anti-CD3 ⁇ (145-2C11), rat anti-CD19 (1D3), anti-CD5 (53-7.3), anti-CD11b (M1/70), and anti-pan NK cells (DX5) for 30 min at 4° C.
  • FITC fluorescein isothiocyanate
  • Cells were then stained with streptavidin-Pe-Cy5 (Dako, Glostrup, Denmark) and sorted as CD11c + Gr1 + CD3 ⁇ ⁇ CD19 ⁇ CD5 ⁇ CD11b ⁇ DX5 ⁇ cells (pDC) using a FACStar plus flow cytometer (Becton Dickinson, Mountain View, Calif.). Sorted cells were washed three times in PBS (Life Technology, Paisley Park, U.K.), resuspended in PBS and frozen at ⁇ 20° C. until time of injection.
  • mAb 120G8 was purified from serum free high density supernatants by anion-exchange chromatography on Hiload Q column (Pharmacia Biotech, Uppsala, Sweden) and coupled with Alexa488 and biotin using standard procedures. Ascites were produced in Balb/c-nude mice (Iffa Credo). Ig isotype was determined by ELISA using a rat Ig subtyping kit (Pharmingen, San Diego, Calif.).
  • 120G8+ cells For surface phenotyping of 120G8+ cells, isolated cells were stained with Alexa-488-labeled 120G8, PE-labelled antibodies (anti-CD3 ⁇ , CD19, DX5, CD11c, CD45R/B220, Ly6C, Ly6G/C (Gr1, RB6-8C5), CD11b, I-A d , H2-K d ) and APC-labelled hamster anti-CD11c (HL-3).
  • BM-DC For staining on BM-DC at the indicated days of culture in FLT3L, cells were stained with 120G8 Alexa488, anti-CD11c-PE, anti-CD11b-PerCpCy5.5 and anti-CD45R/B220-APC.
  • spleen cells maintained in PBS-FCS-EDTA were stained with rat 120G8-Alexa 488, both anti-CD19 and anti-CD3 ⁇ -PerCPCy5.5, anti-CD11c-APC and anti-CD8 ⁇ -PE or anti-CD11b-PE.
  • CD19 + /CD3 ⁇ + cells were gated out using FL3 channel for the analysis. Results are shown as the frequency of the indicated cell subsets among total spleen cells.
  • indicated cells were cultured in complete RPMI1640 medium (at 10 6 cells/ml for non sorted cells and at 0.5 ⁇ 10 6 cells/ml for sorted cells) in the absence or presence of indicated stimuli.
  • the formaldehyde-inactivated human influenza virus, strain NK/TM/138/00 was added to the cultures at a final concentration of 100 hemagglutinin units (HAU) per ml.
  • Phosphorothioate CpG ODNs (TCA TTG GM MC GTT CTT CGG GGC G) (SEQ ID NO: 1) unless otherwise specified, were purchased from MWGBiotech (Munich, Germany) and used at a final concentration of 10 ⁇ g/ml.
  • Recombinant mouse IFN- ⁇ (Hycult Biotechnology, Uden, The Netherlands) was used at the indicated final concentration.
  • Recombinant mouse IFN- ⁇ (R&D) was used at a final concentration of 2 ng/ml.
  • spleen cells were incubated for 30 min at 4° C. with a mixture of mAb including anti-CD3 molecular complex, anti-CD8 ⁇ , anti-CD19 and anti-erythrocyte (TER119) then antibody-coated cells were removed using Dynabeads. Depleted cells were stained with rat 120G8-Alexa488, hamster anti-CD11c (HL-3)-phycoerythrin (PE) for 30 min at 4° C. Cells were then sorted using a FACStar plus flow cytometer (Becton Dickinson), washed and plated in 96-well culture plates with the indicated stimuli for 20-24 h.
  • 120G8 expression after cell activation isolated cells were incubated 20-24 h with the indicated stimuli. Stimulated cells were then stained with 120G8-Alexa488, anti-CD11b-PerCpCy5.5 and anti-CD45R/B220-PE for CD11c+ activated cells or 120G8-Alexa488 and indicated mAbs coupled to PE for total spleen cells. For the latter experiment, indicated cells were gated in using FL2 channel.
  • Spleens were embedded in OCT-compound (Miles) and snap frozen in liquid nitrogen, and stored at ⁇ 80° C. until further analysis.
  • Eight micrometer thick cryosections were fixed in 80% Acetone (Sigma) at ⁇ 20° C. for 20 min, dried at room temperature and stored frozen until stained. Sections were rehydrated in PBS (Life Technology). Avidin/Biotin and peroxydase tissue content were neutralized using specific kit (Vector Laboratory, Burlingame, Calif.) and H 2 O 2 (Sigma) at 0.3% respectively. Sections were blocked with 2% normal mouse serum (Dako, Glostrup, Denmark), and stainings were performed at room temperature.
  • CpG treatment 30 ⁇ l per mouse of the cationic liposome preparation (DOTAP, Roche, Mannheim, Germany) was mixed with 5 ⁇ g CpG ODN in 170 ⁇ l PBS in a polystyrene tube for 10 minutes, before injection into the retro-orbital vein of anesthetized mice. Six hour after CpG injection, spleen were collected and prepared for immunostaining.
  • DOTAP cationic liposome preparation
  • mice were injected i.p. with optimal amount of 120G8 ascites.
  • spleen cells were isolated 24 h after 120G8 injection and stained with anti-Ly6C-FITC, anti-CD45R/B220-PE, anti-CD11c-APC and anti-CD19-PerCpCy5.5 or anti-CD3 ⁇ -PerCpCy5.5.
  • mice were injected i.p. with 120G8 ascites at day ⁇ 1 and at the time of CpG treatment.
  • the antibody was produced from both the parental and the clone 6 in ascites and high density supernatants.
  • MAb 120G8 was found to be of IgG1/ ⁇ isotype as determined by ELISA. As the 120G8+ cells in the spleen appeared to be also CD11c + B220 + Gr1 + cells (formerly defined as mouse pDC) appeared of particular interest, the mAb 120G8 was selected for further studies.
  • 120G8 mAb The reactivity of 120G8 mAb was further examined on unstimulated spleen cells, using double immunofluorescence studies with 120G8 coupled to Alexa 488 and lineage specific markers.
  • 120G8 Ab stained a small subset of freshly isolated splenic cells that was homogenous in forward and side scatter. This subset did not express TER119 (erythrocyte lineage marker), CD19 (B cell lineage marker), CD3 ⁇ (T cell lineage marker) and DX5 (Nk cell lineage marker). All 120G8 + cells were also CD11c low , confirming that the Ab stained a subset of splenic DC.
  • CD11c+ splenic cells have already been demonstrated to be the only cells to produce high amounts of IFN ⁇ in vitro in response to influenza virus (Paturel et al., Nat Immunol, 2001).
  • CD11 c + 120G8 + and CD11c + 120G8 ⁇ cells were purified by flow cytometry from spleen cells depleted of CD3 + CD19 + CD8 ⁇ + TER119 + cells. The two subsets were stimulated in vitro by inactivated influenza virus or CpG as described above. Experiment was performed three times and gave similar results.
  • IFN- ⁇ and IL-12p40 production from both sorted populations incubated with medium alone were below ELISA detection level. Only the 120G8 + subset of CD11c + cells produced IFN- ⁇ after both influenza virus and CpG stimulation. IFN- ⁇ production by 120G8 ⁇ sorted cells in response to both influenza virus and CpG was very low or below detection levels. 120G8+ cells were also able to produce IL-12p40 in response to both stimuli, but for CpG stimulation, at a lower level than 120G8 ⁇ cells, a subset that includes CD11c high DC. This is in agreement with previous data showing that CD8 ⁇ +CD11c high DC are able to produce high amounts of IL-12 in response to various stimuli.
  • Mouse pDC have been previously described to be CD11c+Gr1+B220+ cells in the spleen (Paturel, 2001, Nagano, 2001, Bjorck, 2001), and CD11c+B220+CD11b ⁇ cells in DC derived in vitro from bone marrow cells.
  • CD11c+Gr1+B220+CD11b ⁇ cells in the spleen (Paturel, 2001, Nagano, 2001, Bjorck, 2001), and CD11c+B220+CD11b ⁇ cells in DC derived in vitro from bone marrow cells.
  • 120G8-CD11c+DC CD11c+ ex vivo isolated and in vitro-derived DC in FLT3L were stained with 120G8, CD11c and several Abs coupled to PE.
  • 120G8 also stained previously identified CD11b ⁇ CD11c+B220+ mouse pDC, derived in vitro in FLT3L-stimulated bone marrow cell cultures (BM-DC).
  • BM-DC FLT3L-stimulated bone marrow cell cultures
  • 120G8+ cells were B220 high , Gr1 neg/low , Ly6C high , CD11b ⁇
  • 120G8 ⁇ cells were B220 neg/low , Gr1 ⁇ , Ly6C neg , CD11b + .
  • 120G8 mAb stains both mouse splenic pDC and in vitro derived pDC.
  • 120G8 mAb staining on BM-DC was investigated between day 6 and day 10 of pDC differentiation in vitro.
  • Cells were stained with 120G8, CD11c, CD11b and B220.
  • 120G8 mAb did not stain CD11c ⁇ cells from day 6 to 10.
  • all 120G8+ cells were CD11c+CD11b ⁇ B220+ cells as early as after 6 days of culture.
  • a subset of B220+CD11c+120G8 ⁇ could also be detected, that was CD11b+ and most probably issued from CD11b+ myeloid DC.
  • the staining of 120G8 mAb was investigated in spleen, bone marrow, blood, thymus, peripheral and mesenteric lymph node from Balb/c mice. Isolated cells were analyzed in quadruple surface staining with 120G8, anti-CD45R/B220, anti-Ly6C and anti-CD11c Abs. Ly6C and B220 expressions on CD11c+120G8+ and CD11c+120G8 ⁇ cells was investigated. In all organs tested and in blood, CD11c+120G8+ were all B220 high Ly6C high . In contrast, no CD11c+120G8 ⁇ were B220 high Ly 6 C high . This demonstrate that 120G8 mAb can recognize mouse pDC (CD11c+ B 220 high Ly6C high ) cells, regardless of which tissue they have been isolated from.
  • 120G8 mAb to react with pDC form different mice strains was further investigate.
  • 120G8 mAb reacted with splenic pDC isolated from BALB/cByJ, 129 SvPas, C57BI/6J, CBA/J, C3H/HeN and DBA/2J mice.
  • Spleen cells were isolated from mice of the same age (3 mice per mice strain, experiment performed twice), and stained with 120G8, CD11c, CD19 and CD3 ⁇ . CD19/CD3 ⁇ + cells were gated out for the analysis.
  • mice e.g. the C57/BI6 mice exhibited the lowest splenic pDC frequency (0.6%+/ ⁇ 0.06 of total spleen cells), and the 129Sv mice the highest frequency (1.94%+/ ⁇ 0.37 of total spleen cells).
  • 120G8+ cells The in situ distribution of 120G8+ cells was examined by immunohistochemical analysis of thymus, spleen, peyer's patches, peripheral lymph node, mesenteric lymph node. In all these organs, 120G8+ individual cells could be detected. In some organs (e.g. thymus), some low staining on endothelial cells could be detected. Some low staining on intestinal villosity was also detected.
  • BDCA2, CD123, BDCA4 are three markers commonly used to detect pDC in humans.
  • activated human pDCs down-regulate these markers very rapidly in vitro.
  • it has been to date very difficult to detect activated pDC in situ.
  • the pDC resting or activated
  • this combination stains other cells, e.g. B cells.
  • 120G8 mAb can also stain activated pDC in vivo
  • 129Sv mice were treated with CpG as described in methods.
  • Spleen cells were isolated 6 h after CpG injection, and activation markers, expressed on DC, were studied on DC subsets.
  • 120G8+ cells were upregulating DC activation molecules such as CD40, CD86, and to a lesser extent CD8 ⁇ and MHC class II molecules after in vivo stimulation with CpG. Those cells showed the same level of CD11c (low) and CD11b (negative) in control and CpG treated mice, and the mean fluorescence intensity of 120G8 staining in pDC stayed constant.
  • 120G8 mAb recognizes resting and activated pDC, both in vitro and in vivo.
  • 120G8 mAb stains cell from the marginal zone. No or few 120G8+ cells could be detected in the B or T cell zone. In contrast, a massive influx of CD11c high cells could be detected in the T cell zone.
  • 120G8 mAb can be used to follow pDC migration in response to activation, directly in situ.
  • 120G8 treatment completely abolished IFN ⁇ production induced by CpG treatment (13300 pg/ml+/ ⁇ 1500 for normal mice, less than 150 pg/ml for 120G8 treated mice), while resulting in a small inhibition of IL12 production, both p40 and p70 (IL-12p70:1240 pg/ml+/ ⁇ 540 for normal mice, 300 pg/ml+/ ⁇ 74 for 120G8 treated mice; IL-12p40:5806 pg/ml+/ ⁇ 1135 for normal mice, 4250 pg/ml+/ ⁇ 1170 for 120G8 treated mice).
  • 120G8 mAb can be used to deplete mice from IFN ⁇ producing cells in vivo.
  • 120G8 mAb stains only pDC among total resting cells isolated from normal mice.
  • Spleen cells were isolated from Balb/c mice and incubated 24 h in the presence of cytokines. Double staining with 120G8 and anti-CD19, CD4, CD8 ⁇ or CD11c mAbs was then analyzed on these cells. Autofluorescent cells were gated out using FL3 channel. This demonstrated that the antigen recognized by 120G8 mAb was upregulated on B cells and CD11c+ DC in response to IFN- ⁇ at 100 U/ml, but not on T CD4+ or on T CD8+ DC. This up-regulation was not observed in response to IFN ⁇ , IL-12 or TNF ⁇ . However the mean fluorescence intensity of 120G8 staining on B cells still remained at least one log lower than on pDC.
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